scholarly journals Early detachment of neuromuscular junction proteins in ALS mice with SODG93A mutation

2009 ◽  
Vol 1 (1) ◽  
pp. 16 ◽  
Author(s):  
Hisashi Narai ◽  
Yasuhiro Manabe ◽  
Makiko Nagai ◽  
Isao Nagano ◽  
Yasuyuki Ohta ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Transgenic Animals ◽  
Synaptic Cleft ◽  
Nerve Terminals ◽  
Double Positive ◽  
Copper Zinc Superoxide Dismutase ◽  
Sod1g93a Mice ◽  
The Status

The transgenic animals with mutant copper/zinc superoxide dismutase (SOD1) DNA develop paralytic motor neuron disease resembling human amyotrophic lateral sclerosis (ALS) patients and are commonly used as models for ALS. In the transgenic (Tg) mice with the G93A mutation of the human SOD1 gene (SOD1G93A mice), the loss of ventral root axons and the synapses between the muscles and the motor neurons suggested that the motor neuron degeneration might proceed in a dying-back degeneration pattern. To reveal the relationship between axonal degeneration and the progression of the muscle atrophy in the SOD1G93A mice, we investigated the status of the neuromuscular junction along the disease progression. As a presynaptic or postsynaptic marker of neuromuscular junction (NMJ), anti-synaptic vesicle protein 2 (anti-SV2) antibody and a-bungarotoxin (a-BuTX ) were chosen in this study and, as a marker of synaptic cleft, anti-agrin antibody was chosen in this study. In the immunohistochemistry of a-BuTX and anti-SV2 antibody, the percentages of double positive NMJs among a-BuTX single positive were decreased in Tg mice through time from ten weeks. The number of postsynaptic acethylcholine receptor (AChR) clusters did not decrease in Tg mice even at the end stage. Immunohistochemistry of a-BuTX and anti-agrin antibody revealed that the increase of immunopositive area of anti-agrin antibody around the muscle fiber in Tg mice from ten weeks of age. In this study, we revealed that the detachment of nerve terminals started at ten weeks in Tg mice. The levels of AChR did not change throughout 5-20 weeks of age in both groups of mice, and AChR remains clustering at NMJs, suggesting that the muscle abnormality is the result of detachment of nerve terminals.

scholarly journals A three-dimensional culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction and disease modeling

2018 ◽  
Author(s):  
Mohsen Afshar Bakooshli ◽  
Ethan S Lippmann ◽  
Ben Mulcahy ◽  
Nisha R Iyer ◽  
Christine T Nguyen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Human Skeletal Muscle ◽  
Disease Modeling ◽  
De Novo ◽  
Muscle Fibers ◽  
Three Dimensional ◽  
Adult Human

SummaryTwo-dimensional (2D) human skeletal muscle fiber cultures are ill equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections within two weeks. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium transient imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-culture. This demonstrates that the 3D co-culture system supports a developmental shift from the embryonic to adult form of the receptor that does not occur in 2D co-culture. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. This work delivers a simple, reproducible, and adaptable method to model and evaluate adult human NMJ de novo development and disease in culture.

scholarly journals Tenectin recruits integrin to stabilize bouton architecture and regulate vesicle release at the Drosophila neuromuscular junction

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Qi Wang ◽  
Tae Hee Han ◽  
Peter Nguyen ◽  
Michal Jarnik ◽  
Mihaela Serpe
Keyword(s):  
Neuromuscular Junction ◽  
Motor Neurons ◽  
Synaptic Cleft ◽  
Membrane Skeleton ◽  
Biologically Active ◽  
Striated Muscles ◽  
Local Engagement ◽  
Synaptic Junctions ◽  
Synaptic Boutons ◽  
And Function

Assembly, maintenance and function of synaptic junctions depend on extracellular matrix (ECM) proteins and their receptors. Here we report that Tenectin (Tnc), a Mucin-type protein with RGD motifs, is an ECM component required for the structural and functional integrity of synaptic specializations at the neuromuscular junction (NMJ) in Drosophila. Using genetics, biochemistry, electrophysiology, histology and electron microscopy, we show that Tnc is secreted from motor neurons and striated muscles and accumulates in the synaptic cleft. Tnc selectively recruits αPS2/βPS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and postsynaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. Our study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton.

Tonic Activation of Presynaptic GABAB Receptors in the Opener Neuromuscular Junction of Crayfish

1999 ◽  
Vol 81 (3) ◽  
pp. 1184-1191 ◽  
Author(s):  
Itzchak Parnas ◽  
Grigory Rashkovan ◽  
Jennifer Ong ◽  
David I. B. Kerr
Keyword(s):  
Neuromuscular Junction ◽  
Control Level ◽  
Synaptic Cleft ◽  
Gabab Receptors ◽  
Nerve Terminals ◽  
Pharmacological Profile ◽  
Maximal Inhibition ◽  
Tonic Activation ◽  
Crayfish Neuromuscular Junction ◽  
Excitatory Transmitter

Tonic activation of presynaptic GABAB receptors in the opener neuromuscular junction of crayfish. 1184–1191 Release of excitatory transmitter from boutons on crayfish nerve terminals was inhibited by (R,S)-baclofen, an agonist at GABAB receptors. Baclofen had no postsynaptic actions as it reduced quantal content without affecting quantal amplitude. The effect of baclofen increased with concentration producing 18% inhibition at 10 μM; EC50, 50% inhibition at 30 μM; maximal inhibition, 85% at 100 μM and higher. There was no desensitization, even with 200 or 320 μM baclofen. Phaclofen, an antagonist at GABABreceptors, competitively antagonized the inhibitory action of baclofen ( K D = 50 μM, equivalent to a pA2 = 4.3 ± 0.1). Phaclofen on its own at concentrations below 200 μM had no effect on release, whereas at 200 μM phaclofen itself increased the control level of release by 60%, as did 2-hydroxy-saclofen (200 μM), another antagonist at GABAB receptors. This increase was evidently due to antagonism of a persistent level of GABA in the synaptic cleft, since the effect was abolished by destruction of the presynaptic inhibitory fiber, using intra-axonal pronase. We conclude that presynaptic GABAB receptors, with a pharmacological profile similar to that of mammalian GABAB receptors, are involved in the control of transmitter release at the crayfish neuromuscular junction.

scholarly journals ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction.

1995 ◽  
Vol 130 (6) ◽  
pp. 1423-1434 ◽  
Author(s):  
A D Goodearl ◽  
A G Yee ◽  
A W Sandrock ◽  
G Corfas ◽  
G D Fischbach
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Primary Cultures ◽  
Cholinergic Neurons ◽  
Synaptic Cleft ◽  
Motor Axons ◽  
Amino Terminal

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.

scholarly journals Glia-neuron signaling mediated by two different BMP ligands impacts synaptic growth

2021 ◽  
Author(s):  
Mathieu Bartoletti ◽  
Tracy Knight ◽  
Aaron Held ◽  
Laura M. Rand ◽  
Kristi A. Wharton
Keyword(s):  
Nervous System ◽  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Neural Development ◽  
Growth Function ◽  
Bmp Signaling ◽  
Autocrine Signaling ◽  
Loss Of Function ◽  
Synaptic Growth

ABSTRACTThe nervous system is a complex network of cells whose interactions provide circuitry necessary for an organism to perceive and move through its environment. Revealing the molecular basis of how neurons and non-neuronal glia communicate is essential for understanding neural development, behavior, and abnormalities of the nervous system. BMP signaling in motor neurons, activated in part by retrograde signals from muscle expressed Gbb (BMP5/6/7) has been implicated in synaptic growth, function and plasticity inDrosophila melanogaster. Through loss-of-function studies, we establish Gbb as a critical mediator of glia to neuron signaling important for proper synaptic growth. Furthermore, the BMP2/4 ortholog, Dpp, expressed in a subset of motor neurons, acts by autocrine signaling to also facilitate neuromuscular junction (NMJ) growth at specific muscle innervation sites. In addition to signaling from glia to motor neurons, autocrine Gbb induces signaling in larval VNC glia which strongly express the BMP type II receptor, Wit. In addition to Dpp’s autocrine motor neuron signaling, Dpp also engages in paracrine signaling to adjacent glia but not to neighboring motor neurons. In one type of dorsal midline motor neuron, RP2,dpptranscription is under tight regulation, as its expression is under autoregulatory control in RP2 but not aCC neurons. Taken together our findings indicate that bi-directional BMP signaling, mediated by two different ligands, facilitates communication between glia and neurons. Gbb, prominently expressed in glia, and Dpp acting from a discrete set of neurons induce active Smad-dependent BMP signaling to influence bouton number during neuromuscular junction growth.

scholarly journals Utilizing gene therapy methods to probe the genetic requirements to prevent spinal muscular atrophy.

2016 ◽  
Author(s):  
◽  
Madeline R. Miller
Keyword(s):  
Neuromuscular Junction ◽  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Muscular Atrophy ◽  
Survival Motor Neuron ◽  
Downstream Effects ◽  
Smn Protein ◽  
Progressive Weakness

Spinal Muscular Atrophy is clinically recognized as a progressive weakness within the trunk and proximal limbs that will lead to breathing failure and death within infants. As a neurodegenerative genetic disease, SMA is caused by loss of motor neurons, which in turn is caused by low levels of the Survival Motor Neuron (SMN) protein. The mechanism by which a ubiquitously expressed protein such as SMN is able to cause the specific death of motor neurons is highly debated and of great interest. Work presented here focuses on understanding the biological requirements of SMN and its downstream effects on the neuromuscular junction. To this end we utilize viral based gene delivery as a powerful tool to assess the effects of genes of interest in vivo. Our findings contribute to the conversation regarding whether SMA is truly a "motor neuron" disease, suggesting that astrocytes play a meaningful role in staving off SMA. Further, we investigate the domains within SMN needed to maintain its function in a mammalian system. We take a novel and challenging approach to identify a minimal domain capable of maintaining function. Finally, we demonstrate the practical use of morophological analysis of the neuromuscular junction as a means to characterize SMA pathology.

scholarly journals A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mohsen Afshar Bakooshli ◽  
Ethan S Lippmann ◽  
Ben Mulcahy ◽  
Nisha Iyer ◽  
Christine T Nguyen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Human Skeletal Muscle ◽  
De Novo ◽  
Muscle Fibers ◽  
3D Culture ◽  
Simple Method ◽  
Adult Human

Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.

scholarly journals Modelling functional human neuromuscular junctions in a differentially-perturbable microfluidic environment, validated through recombinant monosynaptic pseudotyped ΔG-rabies virus tracing

2019 ◽  
Author(s):  
Ulrich Stefan Bauer ◽  
Rosanne van de Wijdeven ◽  
Rajeevkumar Nair Raveendran ◽  
Vegard Fiskum ◽  
Clifford Kentros ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Rabies Virus ◽  
Motor Neurons ◽  
Microfluidic Chip ◽  
Neuromuscular Junctions ◽  
Patient Specific ◽  
Human Myotubes ◽  
Virus Tracing

AbstractCompartmentalized microfluidic culture systems provide new perspectives in in vitro disease modelling as they enable co-culture of different relevant cell types in interconnected but fluidically isolated microenvironments. Such systems are thus particularly interesting in the context of in vitro modelling of mechanistic aspects of neurodegenerative diseases such as amyotrophic lateral sclerosis, which progressively affect the function of neuromuscular junctions, as they enable the co-culture of motor neurons and muscle cells in separate, but interconnected compartments. In combination with cell reprogramming technologies for the generation of human (including patient-specific) motor neurons, microfluidic platforms can thus become important research tools in preclinical studies. In this study, we present the application of a microfluidic chip with a differentially-perturbable microenvironment as a platform for establishing functional neuromuscular junctions using human induced pluripotent stem cell derived motor neurons and human myotubes. As a novel approach, we demonstrate the functionality of the platform using a designer pseudotyped ΔG-rabies virus for retrograde monosynaptic tracing.Graphical abstractFunctional neuromuscular junction in a microfluidic chip(a) Overview of microfluidic chip. Human iPS cell-derived motor neuron aggregates (spheroids indicated by black arrows) are seeded in the three lateral compartments of the chip, while human myotubes (white arrows) are seeded in the middle compartment.(b) Directed connectivity and retrograde virus tracing. Outgrowing axons (yellow arrow) from the motor neuron aggregate enter the directional axon tunnels (grey rectangles) and form connections with the myotubes (white arrow) within the opposite compartment. Addition of a designer monosynaptic pseudotyped ΔG-rabies virus to the myotube compartment, infects the myotubes (green) expressing an exogenous receptor (TVA) and rabies glycoprotein (G), subsequently making infectious viruses that are retrogradely transported through the motor neuron axons (green arrow) back to the neuronal cell bodies within the aggregate, validating neuromuscular junction functionality.

scholarly journals Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rowan P. Rimington ◽  
Jacob W. Fleming ◽  
Andrew J. Capel ◽  
Patrick C. Wheeler ◽  
Mark P. Lewis
Keyword(s):  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Unit ◽  
Motor Nerve ◽  
Motor Units ◽  
Extracellular Matrices ◽  
Synaptic Membrane ◽  
Type I ◽  
Dependent Manner ◽  
Human Motor

AbstractInvestigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.

scholarly journals Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Emilia Solomon ◽  
Katie Davis-Anderson ◽  
Blake Hovde ◽  
Sofiya Micheva-Viteva ◽  
Jennifer Foster Harris ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Acetylcholine Receptors ◽  
Gene Networks ◽  
Spinal Cord Injuries ◽  
Regulatory Gene ◽  
Transcriptome Profile

Abstract Background Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. Results Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. Conclusions Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

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